Knowledge has increased concerning the fact that a plant does not act as an individual on its own, but as an actor in a vast stage populated by bacteria, fungi and other microorganisms [1–3]. Arbuscular mycorrhizal (AM) fungi represent one of the most important components of the complex root-plant microbiome, since they are present in about 80% of vascular plants. They supply the plant with phosphate, nitrogen, mineral salts and water, and they guarantee a more extensive protection from biotic and abiotic stresses at both local and systemic level. On the other hand, the plant allows the fungus to access the photosynthetic carbon-compounds .
A partly known chemical dialogue guides this close relationship: plant strigolactones trigger the growth and branching of spore-germinating mycelium, while fungal signals, including lipochitooligosaccharides, stimulate root growth and branching . The latter are recognized by a yet unknown receptor that causes a well conserved signal cascade and induces Ca2+ oscillations within the nuclei, in a similar way to the mechanisms involved in rhizobium-legume symbiosis . Once they have penetrated the epidermis, AM hyphae rapidly develop into plant cortical cells and form a tree-shape structure called arbuscule, which is the functional site of nutrient exchange. Its formation is a non-synchronous process and its life-span is assumed to last no more than 10 days . The arbuscule accommodation process requires a substantial remodelling of the cortical cell: all the thin arbuscule branches are enveloped by a periarbuscular membrane (PAM), which does not simply surround the arbuscule as a whole, but closely follows the surface of each branch, moulding to the arbuscule itself. PAM development marks the appearance of the symbiotic interface, the narrow intracellular compartment that allows AM fungi to grow inside the plant cell without breaking its integrity. Cell biology investigations have recently demonstrated that PAM biogenesis requires the proliferation of the endoplasmic reticulum, Golgi apparatus, trans-Golgi network and secretory vesicles , while the insertion of specific PAM proteins, such as MtPT4, occurs thanks to polarized secretion processes .
All these dramatic events are pointed out by the high number of genes that are regulated, not only in the whole mycorrhizal roots (e.g. [10–13]), but more specifically within the arbusculated cells [14–16]. In this framework, Hogekamp and coworkers have shown that two of the most upregulated membrane transporter genes of Medicago truncatula cells hosting AM arbuscules are aquaporins. This specific gene regulation has also been confirmed by various other reports in different mycorrhizal plants [14, 17–19].
Aquaporins (AQPs) are well known for their ability to transport water, as well as other small solutes (i.e., ammonia, urea, boron), across the membranes of various organisms, and genetic defects involving aquaporin genes have been associated with several human diseases . Plant AQPs are present in various tissues and play a role not just in transport, but also in cell differentiation, cell enlargement, leaf function, nutrient transport and metal toxicity [21–23]. A new database is now available to make enquiries on possible functions through a comparison of sequences and structures .
AQPs are a family of small pore-forming integral membrane proteins. The molecular basis of their selectivity mainly depends on two filters within the pore: the first is formed by the conserved dual “NPA” filters (asparagine, proline and alanine residues), while the second is formed by a constriction region that is also called the ar/R (aromatic/arginine) filter . It appears that the properties of the four residues that make up the ar/R selectivity filter control the substrate specificity of the pore , and are thought to be useful for predicting the function of the protein . On the basis of sequence comparisons, the AQPs of dicots and monocots can be divided into five conserved subgroups, and some of these subgroups appear to be consistently linked to specific subcellular localizations, hence their names: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs), small and basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs) [25, 26]. The overall level of NIP expression in plants is lower than the expression of other AQPs, as they are usually associated with specialized organs and cells, and are involved in the exchange of metabolites between the host and the bacterial symbiont . Among the so far characterized NIPs, AtNIP2;1 specifically accumulates in the endoplasmic reticulum of roots, whereas AtNIP5;1 is a plasma membrane MIP mainly expressed in root elongation zones [28–30]. XIP proteins instead have only been partially characterized, but it seems that they may have various expression patterns and functional characteristics.
Although mycorrhizas have been demonstrated to be crucial for the hydraulic properties of plant roots, as they enhance the tolerance of the host plants to water deficit , the involvement of AQPs in AM symbiosis is still unclear and under debate . It is not known whether the beneficial water status of AM plants is enhanced by the regulation of root aquaporins or because of an enhanced water flow.
With the final aim of shedding light on the potential role of this gene family which seems to be highly AM-responsive, we have focused our research on two AQPs that were found to be upregulated in Lotus mycorrhizal roots , and which belong to the NIP and XIP classes. By means of a combination of different experimental approaches (expression patterns over various nutrient and symbiotic status, transcript localization and quantification of microdissected cells, functional characterization with heterologous assays and GFP-protein localization at a subcellular level through confocal microscopy), we have demonstrated that the two AQPs are genetically and functionally diverse, although they are both AM-responsive. LjXIP1 is exclusively overexpressed in mycorrhizal roots, while LjNIP1 is also Rhizobium responsive. LjNIP1 is solely present in AM roots and, more precisely, in arbusculated cells, where the protein is associated with the complex endomembrane system. Unlike a phylogenetically similar protein , LjNIP1 is involved in water, but apparently not ammonia, transport. These results open new questions on the functional role of AM-responsive AQPs and their relationship with arbuscules as the main fungal colonization structures.